Francisella tularensis is considered a Category A bioweapon due to the ease of transmission, the low infectious dose and high mortality associated with pneumonic tularemia, and the fact that it has been intensively studied and developed in bioweapons programs in several countries. Virtually nothing is known about virulence of aerosolized forms of the most likely bioweapon, F. tularensis subsp. tularensis. The focus of this research effort is to identify all the essential and virulence genes of F. tularensis subsp. tularensis through a genomics based approach. This will dramatically increase our understanding of F. tularensis pathogenesis, and facilitate the development of subunit and live-attenuated vaccines. First, we will utilize a genomics based technique termed GAMBIT to mutagenize every gene of F. tularensis subsp. tularensis, and then recombine them back onto the chromosome. This will allow for the identification of essential genes, which are potential therapeutic targets. The viable mutant bacteria will be combined in pools and inoculated via aerosol into mice, and those mutants that cannot survive within the host will be identified by a microarray-based technique. This technique will identify all genes important for F. tularensis virulence via the aerosol route, some of which may be potential subunit vaccine candidates. Next, the attenuated F. tularensis subsp. tularensis strains will be tested for markers of virulence: ability for intramacrophage survival and growth, resistance to antimicrobial compounds, and ability to invade nonphagocytic cells. This will allow for the identification and characterization of critical virulence determinants of F. tularensis. Finally, we will characterize the virulence regulatory factors identified by microarray analysis of transcription, including the transcriptional activator MgIA, which should illuminate virulence regulons within Francisella, allowing a better understanding of the molecular mechanisms of pathogenesis. Our research plan involves a multidisciplinary approach and high levels of integration with the other projects that compose this program project. Our combined expertise will propel our knowledge of F. tularensis pathogenesis and immunity and allow for the development of novel therapeutic and preventive measures.